>> I THINK BE SHOULD GO AHEAD

AND GET STARTED.

IT'S A PLEASURE TO WELCOME TODAY

SPEAKER RICHARD KOWRS -- VICTOR
GREW UP IN
 A

SMALL COMMUNITY NORTH OF SPAIN

IN A REGION CALLED ASTERIS.

THEY ARE QUITE ISOLATED.

I JUST LEARNED LAST NIGHT OCHOA,

THE 59 NOBLE LAWYE LAUREATE ALSO
CAME

FROM THAT AREA.

VICTOR WAS RECOGNIZED AS A

BRIGHT STUDENT BY HIS TEACHERS

AND WAS INTRODUCED BIOLOGIST AT

THE UNIVERSITY OF MADRID WHERE

HE MATRIC HATED.

HE THEN DID POST DOC TROUBLE

WORK WHERE HE STUDIED REGULATION

AND HEAT GENES.

HE JOINED THE DEPARTMENT OF

BIOLOGY AT JOHNS HOPKINS AS

ASSISTANT PROFESSOR AND

EVENTUALLY BECAME CHAIRMAN OF

THAT DEPARTMENT IN 1998.

IN THE LATE 80'S AND THE 0'S

VICTOR PURSUED A SERIES OF

STUDIES WHICH CHARACTERIZED

MECHANISMS BY WHICH A SPECIAL

CLASS ELEMENTS CALLED CHROMATIN

INSULATORS ALTERED GENE

EXPRESSION.

THESE INLATER ELEMENT INSULATOR
ELEMENTS B
UFFER

GENES -- INCERTAIN BETWEEN THE

ELEMENTS.

THIS GROUND BREAKING WORK

DEFINED A GENERAL MECHANISM BY

WHICH GENETIC INFORMATION IS

ORGANIZED INSULATED BLOCKS OF

EXPRESSION.

ALSO ENLARGED ON THE CONCEPT OF

ACTION AT A DISTANCE BY ENHANCER

ELEMENTS.

SUBSEQUENT WORK IN VICTOR'S LAB

LED TO THE IDENTIFY INDICATION

OF PROTEINS AND FUNCTION OF

THESE ELEMENTS.

AND ALSO SHOWED THAT THEY CAN

COME TOGETHER AT SPECIFIC

NUCLEAR LOCATIONS.

THUS DIFFERENT REGIONS OF THE

GENOME CAN BE ORGANIZED IN

DISTINCT DOMAINS THAT ARE

INDEPENDENTLY REGULATED.

NOW RECENTLY DR. CORCES --

RAPIDLY MOVING TECHNOLOGY ON --

AND I THINK HIS TALK TODAY WILL

FOCUS ON SOME OF THAT WORK.

SO BEFORE VICTOR BEGINS, I'D

LIKE TO MENTION, THERE WILL BE A

RETENTIONION RIGHT OVER HERE IN

THE LIBRARY IMMEDIATELY AFTER

HIS TALK.

SO THOSE OF YOU WHO WOULD LIKE

TO LEARN IN MORE DETAIL PLEASE

COME TO THE RECEPTION.

JOIN ME IN WELCOMING VICTOR.

WE'RE LOOKING FORWARD TO HIS

PRESENTATION.

>> THANK YOU GORDON.

SO AS GORDON HAS ALLUDED TO, I

WOULD LIKE TO DISCUSS THE ISSUE

OF HOW, ARE THE WAY THE

CHROMATIN FIBER IS ORGANIZED,

HOW THAT AFFECTS GENE

EXPRESSION.

MOST OF THE TIME WE'RE USED TO

THINKING ABOUT GENETIC

INFORMATION IN TERMS OF

MODIFICATIONS THAT HAPPEN OF THE

CHROMATIN.

SO WE'RE USED TO THINKING ABOUT

HOW COVALENT -- TO ASK THE

QUESTION OF HOW IS CHROMATIN

ARRANGE IN THE NUCLEUS LEVELS ON

THE NANOMETER FIBER AND HOW THAT

AFFECTS GENE EXPRESSION.

AND ASK THE QUESTION OF WHETHER

THAT ALSO CARRIES THE GENETIC

INFORMATION.

SO IN PARTICULAR, WE ARE

INTERESTED IN WHAT HAPPENS AT

THIS LEVEL, AND I DON'T KNOW

THIS PART OF THE SLIDE IS

DARKER.

I DON'T KNOW IF YOU CAN REALLY

SEE IT OR NOT.

BUT THE CHROMOSOME IN THE

NUCLEUS ARE CONTAINED WITHIN

CHROMOSOME TERRITORY, AND THEN

THE CHROMATIN FIBER IS ARRANGED

IN A CERTAIN WAY IN SOMETHING IF

YOU WERE ABLE TO SEE THIS LIGHT

YOU WOULD THINK IS A PLATE OF

SPAGHETTI.

THE QUESTION IS ARE THOSE

SPAGHETTI AVERAGED I ARRANGED IN
RANDOM

FORM OR A SPECIFIC FORM.

IT CHANGES DURING CELL

DIFFERENTIATION AND IT CARRIES

THE GENETIC INFORMATION BECAUSE

IT ALLOWS THE EXPRESSION OF A

CERTAIN PROGRAM OF TRANSCRIPTION

THAT CAN BE MAINTAINED AFTER THE

CELL DIFFERENTIATES AND AFTER

THE CELL FORMS TISSUES.

SO WHAT IS THE EVIDENCE THAT

CHROMATIN FIBER IS ARRANGED IN A

CERTAIN WAY.

THERE ARE MANY EXAMPLES BUT I

WANT TO SHOW YOU ONE WHICH IS

THE POLY CHROMOSOME

[INDISCERNIBLE]

THIS IS A GOOD EXAMPLE BUT ALSO

THIS LIGHT HAS SPECIAL

SENTIMENTAL MEANING FOR ME

BECAUSE THIS IS THE ONLY SLIDE

THAT I CONTRIBUTED FOR THE WHOLE

TALK.

THIS IS [INDISCERNIBLE] THAT I

DID WHEN I WAS A POST DOC,

PROBABLY -- HELPED ME DO THIS.

BUT ALSO THOSE OF YOU WHO ARE

EXPERTS IN THIS WILL RECOGNIZE

THIS IS THE BEST PAU POLY TIN

CHROMOSOME YOU'VE SEEN IN YOUR

LIFE.

IT'S STAINED WITH THE DNA AND

YOU CAN SEE THERE ARE REGIONS

THAT ARE STAINED DARKLY AND

REGIONS THAT DON'T.

THE DARKLY STAINED REGIONS

PROBABLY HAVE MORE DNA AND THE

LIGHTLY STAINED REGIONS THEY

HAVE LESS DNA.

SO THERE SEEMS TO BE ALTERNATING

PATTERN OF CONDENSED AND

DECONDENSED CHROMATIN IN THE

POLYTIN CHROMOSOMES WHICH ARE 2

TO2,000 PROGRAM SOLES PAIRED
CHROMOSOMES P
AIRED WITH

EACH OTHER.

THINK OF IT NORMAL INSTEAD OF

WHAT THE BEAUTY A POLY CHROMATIN

IS.

LATER IN MY TALK I'M GOING TO

COME BACK TO THIS ARRANGEMENT

AND TO SORT OF SUGGEST THAT IN

DIPLOID CELLS, THE CHROMOSOMES

ARE ALSO ORGANIZED IN THIS WAY.

SO TO DO THAT, I HAVE TO GIVE

YOU A FEW BACKGROUND PIECES OF

DATA.

SO THE FIRST PIECE OF DATA THAT

I WANT YOU TO REMEMBER IS IDEA

THAT CHROMATIN DROSPHILA HAS

FIVE DIFFERENT COLORS.

THIS IS WORK THAT WAS DONE IN

[INDISCERNIBLE] LAB AND WHAT HE

DID WAS MAP THE LOCALIZATION OF

THREE DIFFERENT DROSPHILA

PROTEINS.

TRYING TO FIND SIMILARITIES.

HE CAME UP WITH FIVE DIFFERENT

CLUSTERS THAT HE CALLED GREEN

YELLOW RED BLUE AND BLACK.

AND THOSE DIFFERENT TYPES OF

CHROMATIN WHICH I WILL TALK

ABOUT CONTINUOUSLY IN MY TALK

HAVE SEVERAL CHARACTERISTICS.

SO GREEN IS A PROTEIN THAT HAS

HP1.

IN ADDITION IT HAS SUBAR39 --

AND IT HAS H3 CANINE METHYL TWO.

THOSE ARE THE CHARACTERISTICS IN

GREEN CHROMATIN.

IN GREEN CHROMATIN GENES LOCATED

IN THAT TYPE OF CHROMATIN ARE

EXPRESSED BUT AT LOW LEVELS.

YOU CAN SEE THERE ARE SOME GENES

THAT ARE NOT EXPRESSED BUT THERE

ARE MANY GENES EXPRESSED BUT AT

LOW LEVELS.

NOW YELLOW AND RED CHROMATIN ARE

SLIGHTLY DIFFERENT IN THE TYPE

OF PROTEINS THAT YOU CAN FIND

THERE, BUT IN TERMS OF THE

TYPICAL HISTONE MODIFICATIONS

THAT WE ARE THINKING ABOUT, THAT

WE ARE USED TO THINKING ABOUT,

AK4 TRY METHYL IS WHAT WE

ASSOCIATED WITH ACTIVE CHROMATIN

CHIEFSZ RECHARACTERIZES RED AND
YELLOW

CHROMCHROMATINS.

NOT EVERYTHING IN RED OR YELLOW

CHROMATIN IS TRANSCRIBED.

WHETHER YOU CHROMATIN IS THE

TYPICAL ASSOCIATED WITH POLY

POLYCON -- AND GENES IN BLUE

CHROMATIN ARE IN GENERAL NOT

TRANSCRIBED.

VERY LITTLE IF ANY.

AND THE SAME IS TRUE WITH PLAQUE

PLAQUE -- BLACK CHROMATIN.

IT'S ALSO A REPRESSIVE TYPE OF

CHROMATIN.

IT ENCOMPASSES ALMOST HALF OF

DROSPHILA GENOME.

IT'S BLACK CHROMATIN.

IT'S ALSO REPRESSED.

THE GENES ARE NOT TRANSCRIBED

AND IT HAS A SERIES OF PROTEINS,

SOME OF THEM FAMES PROTEINS LIKE

HISTONE H1 BUT OTHER ONES HAVE

ONLY BEEN STUDIED LIKE D1 FOR

EXAMPLE.

DROSPHILA PROTEIN I'M NOT SURE

IT HAS A HOMOLOGUE.

SO THESE ARE FIVE DIFFERENT

TYPES OF CHROMATIN IN DROI
DROSPHILA

CELLS.

ONE OF THE QUESTIONS I'M REALLY

ADDRESSING IS HOW THE GENETIC

STRUCTURE OF CHROMATIN IN TERMS

OF MODIFICATIONS IN THE TEN

NANOMETER FIBER HOW THAT RELATE

IN THE THREE DIMENSIONAL

ARCHITECTURE OF THE NUCLEUS.

TO ADDRESS THAT QUESTION I'M

GOING TO BE TALKING ABOUT

INSULATORS.

SO INSULATORS ARE PROTEINS THAT

WERE DISCOVERED IN THE 80'S AS

IN1RURINVERTEBRATES, THIS
CHARACTERIZE

I'D INSULATOR WAS AN SF

INSULATOR WAS BORN HERE AT NIH

AND GARY'S GROUP IS RESPONSIBLE

FOR MOST WORK THAT HAS SHED

LIGHT ON WHAT IS INSULATED AND

THE ROLE IN THE CELL.

ANOTHER INSULATOR THAT IS NOW

BEEN STUDIED IN VERTEBRATES IS

THE ONE THAT'S FINED BY THIS

PROTEIN 3F3C AND THIS INSULATOR

WAS ALSO DISCOVERED HERE AT NIH.

SO THIS IS WHAT, THIS IS A VIEW

OF VERT BRATE INSULATORS.

NOW IN DROSPHILA, THERE'S A CTCF

PROTEIN ALSO AND THE CTCF

PROTEIN IN DROSPHILA WAS

CHARACTERIZED BY -- ALSO HERE AT

NIH.

YOU CAN SEE THAT NIH IS THE

BIRTH PLACE OF INSULATORS.

THEY ONLY INSULATE DNA,

OTHERWISE IF THEY INSULATE OTHER

THINGS, YOU PROBABLY DIDN'T NEED

A FENCE, YOU DIDN'T NEED ALL

KINDS OF THINGS.

BUT THEY ONLY INSULATE DNA.

SO CTCF IS THE DROSPHILA

HOMOLOGUE OF THE CTCF PROTEIN.

THEN WE HAVE A WHOLE BUNCH OF

OTHER PROTEINS THAT HAVE WEIRD

NAMES.

TYPICAL DROSPHILA NAMES.

SO SUPPRESSOR -- IS ONE OF THE

INSULATORS.

BEEF IS ANOTHER ONE, GAGA IS

ANOTHER ONE.

WHAT YOU SEE THESE INSULATORS

HAVE IN COMMON IS THE PROTEINS

THAT THEY INTERACT WITH.

SO ALL THIS DNA BINDING PROTEINS

ARE REALLY A WAY FOR THE CELL TO

BRING TO DIFFERENT REGIONS OF

THE JIMMY CERTAIN COMMON GENOME
CERTAIN ON
ES WHICH

ARE CALLED -- AND MODIFIER MCG4.

THAT'S THE INSULATORS IN

DROSPHILA.

IF YOU WERE TO LOOK AT THE

GENOME, YOU WOULD SEE THE

DISTRIBUTION OF THESE PROTEINS

WOULD LOOK SOMETHING LIKE THIS.

SO YOU SEE THESE ARE GENES, SO

THIS GIVES YOU AN IDEA OF HOW

THE GENES ARE ORGANIZED IN THE

DROSPHILA GENOME.

THEY ARE VERY TIGHTLY PACKED,

MUCH MORE SO THAN INVERTEBRATES.

THEN INSULATOR PROTEINS ARE

PRESSING THROUGHOUT.

WHEN YOU FIRST LOOK AT A MAP

LIKE THIS, AT LEAST WHEN WE

FIRST LOOK AT IT, IT'S HARD TO

SEE A SIGNIFICANCE TO IT.

AND WE ARE TRYING TO PUT SOME

ORDER INTO THIS.

BUT FOR THE PURPOSE OF THIS

TALK, WHAT I SHOULD TELL YOU IS

THAT PROBABLY THE INSULATORS

THAT ARE REALLY WORKING IN THE

GENOME ARE THIS SIZE THAT HAVE

ALL THE PROTEINS TOGETHER.

SO YOU CAN SEE FOR EXAMPLE HERE,

CTCF SUPPRESSOR, BELT AF ANBEAF
AND CTCF I
S

HERE WITH SOME OTHER COMPANION

TO MAKE IT ACTIVE.

SO IN A GIVEN CELL, SOME

INSULATORS ARE ACTIVE OR NOT

ACTIVE DEPENDING ON THE PROTEINS

THAT ARE PRESSING ON THE SIDE

AND THERE MUST BE A WAY OF

REGULATING INSULATOR ACTIVITY

THAT IS BASED ON RECRUITING SOME

OF THOSE PROTEINS TO APPROPRIATE

SITES.

AND I'LL COME BACK TO THAT

QUESTION, TO THAT ISSUE TOWARDS

THE END OF THE TALK.

SO AGAIN, WE HAVE CHROMATIN

MODIFICATIONS, PROTEINS IN THE

CHROMATIN THAT DEFINE TYPES OF

CHROMATIN.

WE HAVE INSULATOR PROTEINS THAT,

I'M NOT TELLING YOU ABOUT IT BUT

I'M NOT DESCRIBING IT BUT PEOPLE

HAVE DEMONSTRATED IN DIFFERENT

EXPERIMENTAL SYSTEMS THAT THEY

CAN MEDIATE INTRACHROMOSOMAL

INTERACTIONS.

SO THE QUESTION IS HOW DO THEY

ALL COME TOGETHER AND WHAT THEY

ARE DOING.

SO TO ADDRESS THAT QUESTION WE

ARE USING A METHOD DEVELOPED BY

JOBDECKER THAT ALLOWS YOU TO

INTERROGATE ALL THE INTERACTIONS

THAT TAKE PLACE IN THE GENOMES.

I'M GOING TO GO A LITTLE SLOWLY

THROUGH THE TECHNIQUE AND THE

CHALLENGES SO THAT YOU

UNDERSTAND THE SIGNIFICANCE OF

THE RESULTS.

SO IN HIGH CS YOU HAVE TWO

REGIONS INTERACTING.

YOU DIGESTED DNA WITH A SPECIFIC

RESTRICTION ENZYME.

YOU HAVE A SITUATION LIKE THIS,

SO YOU HAVE TO CROSS LINK THE

DNA AND THE PROTEINS FIRST.

THEN YOU FILL IT IN WITH

NUCLEOTIDES THAT HAVE B BIOTIN.

YOU DO THE CROSS LIKE AND YOU

THEN ISOLATE THE SPECIFICALLY

THE FRAGMENTS THAT HAVE THE

BIOTIN WITH THE -- COLUMN AND

THEN YOU CAN DO DEEP SEQUENCING

OF FRAGMENTS.

SO WHAT YOU GET IS VERY LARGE

COLLECTION SEQUENCES.

SO WE'VE DONE BIOLOGICAL

REPLICATE AND TECHNICAL

REPLICATE.

THERE'S HIGH CORRELATION BETWEEN

THE DATA AND WE ENDED POP WITH

82 MILLION PAIR AFTER WE REMOVED

ALL THE THINGS THAT WERE BOUGHT

ACCORDING TO THE STATISTICAL

PEOPLE THAT, THE STATISTICS

PEOPLE THAT HELP US ANALYZE THIS

DATA.

SO WHAT DO YOU DO WITH 82

MILLION READS.

SO TO PUT THIS INTO CONTEXT, IF

YOU'RE FAMILIAR WITH THE PAPER

THAT JOB DECKER PUBLISHED ON THE

THREE DIMENSIONAL ARCHITECTURE

OF HUMAN CHROMOSOMES AND HUMAN

CELLS.

I BELIEVE THAT IN THAT CASE YOU

ANALYZE AROUND NINE MILLION

READS.

THE HUMAN GENOME IS BIGGER THAN

DROSPHILA AND THE RESOLUTION

THAT THEY COULD REACH IN THAT

CASE WAS AROUND ONE MEGA BASE.

IN OUR CASE WITH THE DROSPHILA

GENOME AND THIS NUMBER OF READS,

WE CAN REACH A RESOLUTION OF A

SINGLE FRAGMENT WITHIN A

SPECIFIC TWO MEGA BAY REGIONS.

SO WE HAVE SINGLE FRAGMENT

RESOLUTION.

SO WHAT DO WE SEE, THAT'S WHAT

WE SEE MAKES SENSE.

SO THIS IS A CONTACT MATRIX OF

THE INTERACTIONS ALONG ALL THE

CHROMOSOMES.

SO I'M SHOWING YOU THIS PICTURE

FIRST OF ALL TO SHOW YOU THAT

THERE ARE THESE REGIONS OF

INTERACTIONS THAT FORM THESE

MODULES LIKE SQUARES, AND I'M

GOING TO COME BACK TO THIS OVER

AND OVER.

AND THIS MODULE FLYING THE

DIAGONAL AND THE DIAGONAL MEANS

INTERACTIONS WITH THE FRAGMENT

WITH ITSELF IS PRESENT.

SO WE ELIMINATE THOSE READS.

BUT WHAT IS INTERESTING IN THIS

LIFE IS THAT YOU CAN SEE, ARE

THIS IS THE CHROMATIN OF

CHROMOSOME TWO AND IT INTERACTS

WITH ITSELF BECAUSE THIS IS THE

LEFT ARM AND THIS IS THE RIGHT

ARM OF CHROMOSOME TWO.

SO YOU CAN SEE IT INTERACTS WITH

CHROMOSOME THREE AND WITH

CHROMOSOME X AND WITH CHROMOSOME

4.

AND WHAT THIS MEANS IS THAT THE

CENTROMERES OF THE CHROMOSOMES

ARE COMING TOGETHER.

THEY ARE INTERACTING WITH EACH

OTHER.

AND THIS IS KNOWN TO HAPPEN IN

DROSPHILA.

IT ALSO HAPPENS IN HUMAN CELLS.

AND MORE OR LESS DEPENDING ON

THE CELL LINE.

BUT IF YOU REMEMBER THE POLY TIN

CHROMOSOMES I SHOWED YOU

BEFORE -- IN A DIPLOID CELL THE

CHROMOSOMES ARE ALSO INTERACTING

TOGETHER.

THE OTHER THING THAT YOU SEE IS

THERE ARE VERY FEW, THERE ARE

SOME INTERACTIONS AND YOU SEE

MORE WHEN I FOCUS ON THE

SPECIFIC CHROMOSOMES.

BUT CHROMOSOME 2L DOESN'T

INTERACT WITH CHROMOSOME 2R MORE

THAN IT INTERACTS WITH 3L OR 3R.

AND SO WHAT THAT MEANS IS THAT

THE CHROMOSOMES IN THIS SPECIFIC

CELL LINE, THEY ARE NOT IN A --

IT'S NOT LIKE THE TWO ARMS OF

THE CENTROMERES ARE ONE END.

THE CHROMOSOMES ARE ORGANIZED IN

A DIFFERENT WAY.

SO IN ORDER TO ANALYZE THE DATA,

WE DID THE FOLLOWING.

SO THIS IS A REGION OF THE

GENOME IN WHICH WE MAP 500

FRAGMENTS.

SO THIS IS AROUND 200 MEGA

BASES.

I'M SORRY FOR GOING INTO THE

TECHNICAL DETAILS BUT IT'S

IMPORTANT IN ORDER FOR YOU TO

UNDERSTAND IF WHAT I'M TELLING

YOU IS TRUE OR NOT.

SO AGAIN, YOU SEE THESE REGIONS

OF HIGH INTERACTIONS, AND THEN

OTHER REGIONS OF LOWER

INTERACTIONS THAT SORT OF

OVERLAP WITH THE REGIONS OF LOW

INTERACTIONS.

SO IF WE TAKE ALL THE READS AND

WE LOOK AT THE FREQUENCY OF

INTERACTION WITH RESPECT TO THE

DISTANCE AND WE MEASURE THE

DISTANCE NOT IN KB BUT IN

FRAGMENT NUMBERS, THE BLUE LINE

HERE IS THE AVERAGE OF ALL THE

GENOME.

NOW, IF WE CONSIDER JUST THIS

MODULES, I'M GOING TO CALL THEM

CHROMOSOME MODULES FROM NOW ON,

YOU SEE THAT THE INTERACTION

DECREASES SLOWLY AND THEN

THERE'S A POINT WHERE IT

DECREASES VERY FAST.

AND THEN IT DECREASES SLOWLY

AGAIN.

THIS POINT WHERE THE INTERACTION

GOES DOWN IS THE REGION IN

BETWEEN TWO MODULES, AND WE'LL

CALL THAT INTERCHROMOSOMAL

MODULE.

INTERMODULE REGION.

SO WHAT WE'VE DONE IS SORT OF

MEASURE, CALCULATE A MODULARITY

INDEX THAT IS A WAY OF

CALCULATING THIS ESSENTIALLY.

AND THEN ARBITRARILY WE CUT OFF,

WE MADE A CUT OFF AT POINT SIX.

SO ANYTHING THAT HAS MORE THAN

POINT SIX MODULARITY INDEX IS A

MODULE, ONE OF THESE AND

ANYTHING THAT HAS LESS THAN THAT

IS AN INTERMODULE.

SOMETHING LIKE THAT, OKAY.

SO IF YOU THINK ABOUT THE WAY

THIS REGION OF THE CHROMOSOME IS

ORGANIZED, YOU HAVE AN

INTERMODULE REGION THAT IS LIKE

A LINE AND THEN A MODULE WHERE

THE CHROMATIN IS MORE CONDENSED,

AND THEN ANOTHER INTERMODULE

REGION AND THEN ANOTHER MODULE

WHERE THE CHROMATIN IS MORE

CONDENSED.

NOW REMEMBER, THE REASON WHY

IT'S CONDENSED IS BECAUSE OF

THIS INTERACTIONS.

AND THIS INTERACTIONS WE'VE

ELIMINATED ALL SHORT RANGE

INTERACTIONS.

SO PROBABLY WE'RE NOT TAKING

INTO ACCOUNT THINGS LIKE THE 39

NANOMETER FIBER.

THESE INTERACTIONS ARE NOT

TAKING INTO ACCOUNT INTERACTIONS

THAT WOULD HAPPEN AS A

CONSEQUENCE OF THE CHROMATIN

BEING IN THE FORM OF THE

NANOMETER FIBER.

SO WHEN WE CALCULATE THIS

MODULARITY INDEX, THEN, FOR

EXAMPLE HERE, WE WOULD CALL THIS

REGION AN INTERMODULE AND IT

CORRESPONDS TO THIS.

IN THE HEAT MAP, THIS IS ANOTHER

INTERMODULE.

THIS IS A MODULE.

SO BY DOING THIS THROUGH THE

GENOME, WE CAN SUBDIVIDE THE

GENOME INTO MODULES AND

INTERMODULES.

AND IF YOU LOOK AT THE COLORS OF

THE CHROMATIN, THIS IS THE

COLORS OF THE CHROMATIN THAT I

MENTIONED BEFORE, YOU CAN SEE

THAT IN GENERAL, THERE'S A

PRETTY GOOD CORRELATION BETWEEN

A MODULE AND THE TYPE OF

CHROMATIN THAT IS IN IT.

SO FOR EXAMPLE IN THIS MODULE IS

ALL REPRESSIVE CHROMATIN.

EITHER BLACK OR BLUE.

IN THIS MODULE HERE, IT'S EITHER

YELLOW OR RED.

IT'S ALL ACTIVE.

THE SAME HERE.

SO THERE'S NOT A PERFECT

CORRELATION, BUT THERE IS A

PRETTY GOOD CORRELATION BETWEEN

THE DEFINITION OF A MODULE AND

THE FACT THAT THAT MODULE HAS

ONE TYPE OF CHROMATIN AS DESIGN

OF FINED BY EPIGENETIC

MODIFICATIONS.

SO THIS GIVES YOU SORT OF A

GENOME-WIDE VIEW OF THIS, OF

WHAT I JUST SAID.

SO THE AVERAGE THE GENOME

AVERAGE FOR THE DIFFERENT TYPES

OF CHROMATIN IS HALF OF THE

GENOME IS BLACK AND THEN A BIG

CHUNK OF IT IS BLUE.

SO A LOT OF THE GENOME IS

REPRESSED, AND THEN A THIRD OF

THE GENOME IS ACTIVE.

WHEN WE LOOK AT THIS CHROMOSOME

MODULES, WE'VE SEEN CHROMOSOME

MODULES, THE DISTRIBUTION IS

MORE OR LESS THE SAME.

WHEN WE LOOK AT THE NUMBER OF

MODULES IN THE DIFFERENT

REGIONS, WE SEE SOMETHING THAT

IS NOT VERY DIFFERENT.

SO WHAT THIS TELL US IS THAT

THERE ARE A LOT OF CHROMOSOME

MODULES, A THIRD OF ALL OF THEM

HAVE BLACK CHROMATIN REPRESSIVE,

SIX OF THEM HAVE BLUE CRIME

CONTINUE, POLY CHROME CHROMATIN

AND THEN ANOTHER THIRD.

I DIDN'T GET ALL MY THIRDS

RIGHT.

ALL HAVE ACTIVE CRIME CONTINUE.

OKAY.

SO THAT'S THE DISTRIBUTION OF

CRIME CONTINUE WITHIN THE

MODULES.

THE MODULE SIZE ON AVERAGE IS

AROUND, IS NOT REALLY INFLUENCED

BY THE CHROMATIN SO THIS HAS

DIFFERENT COLORS OF CHROMATIN.

IT'S AROUND, SOMEWHERE AROUND 50

TO 100 KILO BASES BUT THERE ARE

MODULES UP TO 500 KILO BASES

LONG.

OKAY.

AND WHEN WE LOOK AT THE GENE

DENSITY IN THE MODULE REGIONS,

THEY HAVE ON AVERAGE, AND THIS

IS ALSO INDEPENDENT OF THE TYPE

OF CHROMATIN, AN ARRANGE OF ONE

GENE PER 10KB.

WHEN WE LOOK AT THE

TRANSCRIPTION RATE OF GENES IN

MODULES, WE SEE THAT THERE ARE

REPRESSED GENES IN ALL OF THEM

AND THERE ARE ACTIVE GENES IN

ALL OF THEM.

AND IT DOESN'T DEPEND ON THE

EPIGENETIC TYPE OF CHROMATIN.

SO LET ME SAY THIS AGAIN BECAUSE

THIS IS THE ONLY IMPORTANT THING

OF MY TALK.

AFTER THIS, YOU CAN LEAVE.

WE HAVE A REGION, REGIONS IN THE

GENOME THAT FORM THESE THINGS

THAT WE CALL MODULES.

THESE REGIONS HAVE HIGHER LEVEL

OF INTERACTIONS COMPARED TO

NEIGHBORING REGIONS.

I HESITATE TO SAY CONDENSED

CHROMATIN.

THEY ARE JUST INTERACTING MORE

FREQUENTLY.

THESE REGIONS ARE NOT VERY RICH

IN GENES, BUT THE GENES THAT ARE

PRESENT, THE GENES THAT ARE

PRESENT IN THESE REGIONS ARE

TRANSCRIBED OR NOT TRANSCRIBED

INDEPENDENT OF THE TYPE OF

HISTONE MODIFICATION OR THE TYPE

OF CHROMATIN THAT THEY HAVE.

SO THIS REGIONS ARE TRANSCRIBED

MORALS WHETHEMORE OR LESS
WHETHER THEY HAV
E

H3K TRY METHYLATION OR HK27 TRY

METHYLATION.

WHAT I'M TRYING TO ARGUE IS WHAT

IS IMPORTANT FOR THE

TRANSCRIPTION IS THE FACT THAT

THEY ARE IN A MODULE.

NOW WHAT HAPPENS TO THE

INTERMODULE REGIONS?

THE INTERMODULE REGIONS THE TYPE

OF CHROMATIN IS MORE OR LESS THE

SAME.

A THIRD IS BLACK, A THIRD IS

GREEN OR RED SO THERE'S NOT THAT

DIFFERENT IN THE TYPE OF

CHROMATIN BETWEEN THE

INTERMODULES AND MODULES AND THE

NUMBER OF THEM IS MORE OR LESS

AS THE MODULE.

THE SIZE IS MUCH SMALLER.

THE AVERAGE SIZE OF THIS

INTERMODULE REGIONS IS 20 KILO

BASES SO THEY'RE NOT JUST MORE

THAN THE MODULES.

THEY ARE SLIGHTLY, THEY HAVE

MORE GENES.

THEY HAVE AROUND TWO GENES PER

10KB.

SO THE GENE DENSITY IN THESE

REGIONS IS HIGHER.

AND THEY ARE TRANSCRIBED MORE.

AND THEY ARE TRANSCRIBED MORE IN

THIS CASE, THE TYPE OF CHROMATIN

HAS AN EFFECT.

SO FOR EXAMPLE IN BLACK

CHROMATIN, THERE ARE MORE GENES

THAT ARE COMPLETELY REPRESSED

THAN IN RED OR YELLOW

CHROMOSOME.

CRIMCHROMATIN.

BUT AGAIN THE TRANSCRIPTION AS

YOU REMEMBER FROM BEFORE, THE

TRANSCRIPTION LEVEL OF GENES IN

INTERMODULE IS HIGHER THAN IN

MODULES, EVEN FOR THE SAME TYPE

OF CHROMATIN.

SO AGAIN, THE INTERMODULE

REGIONS ARE REGIONS THAT DON'T

HAVE INTERACTIONS.

SO YOU COULD CALL THIS A MORE

OPEN CHROMATIN.

WHAT IS DETERMINED IN THE

TRANSCRIPTION LEVEL IS NOT THE

TYPE OF CHROMATIN IS THE FACT

THAT THEY ARE IN INTERMODULES.

NOW, WHAT DO WE FIND IN THIS

DIFFERENT REGIONS, WHAT KIND OF

PROTEINS DO WE FIND THAT ARE

INTERESTING?

SO THE INTERMODULE REGIONS ARE

DEPLETED FOR PROTEINS OF BLACK

CHROMATIN, BLUE CHROMATIN AND

GREEN CHROMATIN.

AND THEY ARE, THEY HAVE AN OVER

REPRESENTATION OF INSULATOR

PROTEIN, OKAY.

AND THE INSULATOR PROTEINS WERE

PARTIALLY USED BUT NOT REALLY

USED TO DEFINE THESE TYPES OF

CRIME CONTINUE.

 TYPE -- CHROMATIN.THESE ARE
SPECIFIC FOR 
BLUE

CRIME CONTINUE.

HP1SUBAR ARE SPECIFIC FOR THREE

CHROMATIN.

BUT INSULATOR PROTEINS DON'T

CARE ON THE TYPE OF CRIME

CONTINUE.

THEY ARE PRESENT IN ALL TYPES OF

CHROMATIN BUT THEY ARE

PREFERENTIALLY PRESENT AT

INTERMODULES.

OKAY.

SO LET'S LOOK AT THESE MODULES

AND INTERMODULES IN A LITTLE

MORE DETAIL.

SO WE USE A PROGRAM OR AN AL --

ALGORITHM WE'VE BEEN

COLLABORATING WITH -- AT HARVARD

AND THEY DEVELOPED SOMETHING

CALLED -- 3D CONSTRUCT FOR DATA.

THIS PROGRAM IS ABLE TO TAKE THE

INTERACTIONS AND RECONSTRUCT A

3-DID DIMENSIONAL MODEL BASED ON

THE 3-D INTERACTIONS.

SO WE CAN LOOK AT A PICTURE OF

THE HEAT MAP, OKAY.

SO HERE WE HAVE A REGION THAT IS

80 KILO BASES.

AND IT HAS A SMALL MODULE HERE

OF, A SMALL MODULE HERE, TWO

MODULES HERE.

AND THEN SEVERAL INTERMODULE

REGIONS.

IF WE TAKE INTO ACCOUNT ALL

THESE INTERACTIONS AND WE TRY TO

REPRESENT THREE DIMENSIONAL

MODEL OF WHAT THAT WOULD LOOK

LIKE BASED ON THESE

INTERACTIONS, THIS IS WHAT IT

LOOKS LIKE.

THIS IS AT LEAST WHAT THIS

ALGORITHM TELLS US THAT IT LOOKS

LIKE.

SO YOU CAN SEE THAT THE CRIME

CONTINUE IS FOLDING BUT IT'S NOT

COMPACTED IN THE WAY WE ARE USED

TO THINKING ABOUT COMPACTED

CHROMATIN.

IF NOW WE ZOOM OUT AND WE LOOK

AT A BIGGER REGION, NOW WE SEE

SOMETHING LIKE THIS.

SO THIS REGION IS NOW LOCATED IN

HERE.

SO AS WE ZOOM OUT, BECAUSE WE

DON'T HAVE ENOUGH READ, WE LOSE

RESOLUTION.

SO ALTHOUGH HERE WE HAVE SINGLE

FRAGMENT RESOLUTION IN THESE

OTHER PICTURES WE DON'T HAVE

SINGLE FRACTMEN FRAGMENT
RESOLUTION.

SO WE LOSE RESOLUTION.

WE'RE NOT ABLE TO SEE THE DETAIL

BUT WE KNOW THIS IS REPRESENTED

BY THIS.

IF WE GO EVEN FARTHER OUT, THIS

IS HALF OF THE CHROMOSOME, AGAIN

YOU CAN SEE THIS WHOLE REGION

HERE IS REPRESENTED BY THIS KINK

HERE AND YOU CAN SEE HOW THIS

HALF OF THE CHROMOSOME IS FOLDED

IN THE CD SPACE.

NOW, AN IMPORTANT POINT HERE IS

THAT THIS REGION THAT IS 80 KILO

BASES IS JUST CONTAINED IN HERE.

AND YOU SEE IN THIS REGION OF

THE CHROMOSOME, WE HAVE THIS

MODULE, ANOTHER ONE ANOTHER ONE

AND THEN THERE'S AN OVER-ARCHING

MODULE THAT COVERS THEM ALL.

OKAY?

SO THIS STRUCTURE THAT WE CALL A

LARGE MODULE, LARGE CHROMOSOME

MODULE MUST FORM BY INTERACTIONS

IN BETWEEN SEQUENCES WITHIN IT,

WHICH MEANS SEQUENCES BETWEEN

THE INDIVIDUAL MODULES THAT I

MENTIONED BEFORE.

AND AS YOU ZOOM OUT TO A LARGER

PLACE IN THE CHROMOSOME, THIS

WHOLE REGION IS NOW IN HERE AND

YOU CAN SEE THAT THIS REGION IS

NOW WITHIN A BIGGER MODULE.

SO THE CHROMOSOME IS ORGANIZED

IN THIS HIERARCHICAL STRUCTURE

IN WHICH IF WE START FROM THE

WHOLE CHROMOSOME HAS MODULES AND

INTERMODULES.

BUT IF WE GO LOOK IN ONE OF

THOSE MODULES, THERE ARE SMALLER

MODULES AND INTERMODULES.

AND IF WE LOOK ON EACH OF THEM,

THERE'S EVEN A SMALLER ONE.

I'M NOT SURE WHETHER I'M MAKING

MYSELF CLEAR WITH THIS STATEMENT

BECAUSE I DON'T HAVE A PICTURE.

BUT MAYBE YOU CAN VISUALIZE THAT

IN YOUR HEAD.

SO THIS LARGE CHROMOSOME MODULES

AND INTERMODULES, THEY HAVE

SIMILAR PROPERTIES AND I WON'T

GO THROUGH THEM BECAUSE THEY ARE

NOT IMPORTANT.

SO I TOLD YOU THAT THE LARGE

MODULES AND THE INTERMODULES

ARISE FROM INTERACTIONS AMONG

THE SMALL ONES.

SO IN THIS PICTURE, YOU CAN SEE

WHAT I MEAN.

SO IN THIS PICTURE, WE HAVE A

SMALL MODULE, ANOTHER ONE HERE

AND ANOTHER ONE HERE.

AND SO HERE AND HERE, WE CAN SEE

INTERACTIONS THAT ARE HAPPENING

BETWEEN THIS INTERMODULE REGION

AND THIS INTERMODULE REGION.

SO WHAT MAKES THE MODULES

FORM -- ARE THE INTERACTIONS

BETWEEN THE MODULES AT A LOWER

LEVEL.

REALLY WHAT THEY ARE IS

INTERACTIONS BETWEEN THE BORDERS

OF THE MODULES ALTHOUGH YOU

CAN'T APPRECIATE IT HERE.

THE INTERACTIONS ARE BETWEEN THE

BORDERS OF THE MODULES.

SO AGAIN, TO ILLUSTRATE THIS

AGAIN, IN REGION FALLS IN

SOMETHING LIKE THIS.

SO THIS BIG MODULE IN HERE IS

THIS REGION HERE IN THE MIDDLE.

SO YOU CAN GET AN IDEA OF HOW

IT'S FOLDING, SO A MODULE IS

MORE COMPACTED, IT'S NOT

COMPACTED AS COMPACTED AS THE 30

NANOMETER FIBER FOR EXAMPLE.

SO WHAT IF THESE INTERACTION

SITES THAT I JUST TOLD YOU

ABOUT, THESE SITES THAT ARE

LOCATED AT THE BORDERS BETWEEN

MODULES AND INTERMODULES, AND

ARE INTERACTING WITH EACH OTHER

TO FOLD THE CHROMOSOME INTO

HIGHER LEVELS OF ORGANIZATION.

WHAT IS THERE.

SO WHEN WE EXAMINE THIS GENOME

WIDE, YOU CAN SEE THIS IS A META

MODULE.

AND THESE ARE INSULATOR

PROTEINS.

THE ONLY PROTEIN THAT WE SEE A

CORRELATION BETWEEN THE

INTERACTION SITES AND THE

PROTEIN SITE IS INSULATOR

PROTEINS.

SO YOU CAN SEE INSULATOR

PROTEINS ARE THROUGH THE REGION

BUT THESE BOUNDARIES ARE

INTERACTING WITH EACH OTHER ARE

MORE ENRICHED.

THE OTHER THING THAT IS MORE

ENRICHED RIGHT THERE AFLT THESE

BOUNDARIES IS HIGHLY EXPRESSED

GENES.

HIGHLY EXPRESSED GENES MEAN RNA

POLYMERASE.

IN THESE ANALOGIES WE CAN

ONLY -- WE CAN'T DECIDE ON CAUSE

AND EFFECT.

SO I'M QUICKLY GOING TO FLIP

THROUGH SOME OF THE SLIDES TO

SHOW YOU WHAT THIS LOOKS LIKE.

SO WHAT I'M GOING TO SHOW YOU

NOW IS A SUBSET OF INTERACTIONS

THAT ARE WHAT WE CALL THE LONG

RANGE INTERACTIONS.

SO THEY ARE THE INTERACTIONS

BETWEEN THESE MODULE INTERMODULE

BOUNDARIES THAT ARE FOLDING THE

CHROMOSOME.

THEY ARE NOT THE INTERACTIONS

WITHIN THE SMALL MODULES, OKAY.

AND THE PICTURES I'LL SHOW YOU

ARE FOR CHROMOSOME 2R.

SO THESE ARE ALL THE

INTERACTIONS AT THE SINGLE

FRAGMENT LEVEL OF RESOLUTION

THAT WE CAN SEE IN CHROMOSOME

2R.

SO YOU CAN SEE THAT THERE ARE

MANY INTERACTIONS.

THAT THERE'S A HUGE METAL MESS,
AND

THAT'S GOING TO TAKE A LONG TIME

TO FIGURE OUT WHAT THEY ARE.

NOW, IF WE TRY TO BREAK THEM

INTO CATEGORIES, THESE ARE THE

INTERACTIONS NEEDED BY INSULATOR

PROTEINS.

SO IN THIS DIAGRAM, THESE ARE

THE DIFFERENT TYPES OF CRIME

CONTINUE.

YOU PUT DIFFERENT COLORS.

THESE ARE THE LOCATIONS OF THE

DIFFERENT INSULATOR PROTEINS I

DO REMEMBER BUT THEY ARE CTCF

AND CP190.

AND THEN THESE ARE THE

INTERACTIONS.

AND IF YOU WERE TO COME UP HERE

CLOSE TO THE SCREEN AND LOOK AT

THESE INTERACTIONS, YOU WOULD

SEE THAT THE INTERACTING SITES

ARE LOCATED WHERE THE PROTEINS

ARE TOGETHER IN THE CHROMOSOMES.

THEY DO NOT LOOK AT THEM

TOGETHER THEN THERE'S NO

INTERACTION.

THIS IS WHAT I MENTIONED AT THE

BEGINNING.

IF WE LOOK AT THE SITES OF THESE

LOOPS THAT ARE FORMED, INSULATOR

TENDS TO FORM LOOSE.

THEY DON'T REALLY CARE ABOUT THE

SITES.

THEY TEND TO FORM LOOPS THAT GO

FROM 50KB ALL THE WAY TO 4 MEGA

BASES.

SO THEY CAN FORM RELATIVELY

SHORT INTERACTIONS UP TO FOUR

MEGA BASES.

THESE ARE THE INTERACTIONS THAT

ARE MEDIATED BY POLY CON.

IF YOU LOOK ALL THE INTERACTIONS

ARE ME CREATED BY GREEN

CHROMATIN.

THESE ARE SEVERAL POLYCON GROUP

PROTEINS.

POLYCON THE INTERACTIONS CLUSTER

INTO TWO DIFFERENT SIZES.

ONE IS AROUND ONE MEGA BASS AND

THE OTHER IS AROUND THREE MEGA

BASE.

WHAT THAT MEANS IS SOME OF THE

INTERACTIONS ARE LOCAL AND BRING

TOGETHER ONE OR TWO POLYCON TYPE

DOMAINS.

AND THE OTHER INTERACTIONS ARE

LONG RANGE AND ARE BRINGING

TOGETHER POLY CONS THA PAUL.

>TOGETHER.

>TOGETHER POLY CON.

THIS IS THE CHROMATIN IN

CHROMOSOME 2R.

AND THEN FORM A FEW LOOPS WITH

GREEN CHROMATIN WHERE HP1 IS

PRESENT THROUGH THE

 CHROMATIN

AND MOST OF THE INTERACTION ARE

DEFINITE SIZE AROUND I THINK THE

SCALE IS WRONG.

AROUND 500KB ARE GOING TO THIS.

FINALLY, THESE ARE INTERACTIONS,

NOT FINALLY BUT MEDIATED BY

LAMIN.

YOU CAN SEE GREEN CHROMATIN IS

VOID LAMIN.

THERE'S NO INTERAIONS.

THIS IS NOT PRESENT IN THE

LAMIN.

THESE ARE THE REGIONS THAT'S

MAPPED AND IF YOU LOOK YOU CAN

SEE THAT MOST OF THE

INTERACTIONS ARE REALLY NOT

MEDIATED BY THE ACTUAL REGION,

BY THE ACTUAL LAMIN ASSOCIATED

DOMAIN BUT THEY ARE MEDIATED BY

THE BOUNDARIES, THE BORDERS OF

THAT DOMAIN.

AND THE PEAKS CLUSTER INTO SMALL

AND LARGE MAKES AGAIN JUST LIKE

POLYCON.

AND THIS IS WHAT RNA POLYMERASE.

THEY ARE MOST LOCATED IN YELLOW

OR RED CHROME CONTINUES.

SO MANY OF THEM ALSO IN GREEN

CHROMATINS.

A LOT OF THEM COINCIDE WITH

INSULATOR PROTEINS.

SO THAT'S AN OVERLAP BETWEEN

LOOPS MEDIATED BY RNA POLYMERASE

AND LOOPS MEDIATED BY INSULATOR

PROTEINS.

AND IF YOU WANT TO SPECULATE,

YOU MACON CLUDE FRO MAY CONCLUDE
FROM THIS
 IS

ONE OF THE THING THE INSULATORS

ARE DOING IS BRINGING

TRANSCRIBED GENES TOGETHER IN A

REGION OF THE NUCLEUS THAT COULD

BE TRANSCRIPTION FACTORY.

SO I'M GOING TO SKIP SOME OF

THIS INFORMATION BECAUSE I HAVE

ALREADY TOLD YOU.

SO LET ME TELL YOU I THINK ACUTE

RESULT.

ARE YOU GUYS STILL WITH ME?

I DIDN'T BORE YOU COMPLETELY?

SO MANY OF YOU ARE FAMILIAR WITH

THE FACT THAT DROSPHILA

CHROMOSOMES IN INTERFACE, THE

TWO HOMOLOGUES ARE PAIRED.

THIS DOESN'T HAPPEN -- WE

WONDERED IF WE COULD ACTUALLY

SEE THIS IN THE DATA.

SO THE CHROMOSOMES MAY PAIR

BECAUSE EITHER THEY ARE SPECIFIC

REGIONS THAT ARE PAIRING AND

THAT CAUSES A CHROMOSOME TO PAIR

OR THERE ARE LOT OF REGIONS OR A

COMBINATION OR THE WHOLE ARM IS

PAIRED.

BUT IN THE PROCESS OF ALLIESING

THE HIGH C RESULTS, WE SAW A LOT

OF FRAGMENT IN WHICH A LOT OF

LALIGATION EVENT IN WHICH IF WE

CALL THIS DIRECTION IN ORDER AND

THIS DIRECTION IN ORDER, WE SAW

LIGATIONS IN WHICH WE HAD A

FORWARD LIGATED TO A FORWARD.

WITHOUT GOING INTO A LOT OF

DETAILING AND WHY

 YOU CAN

EXPLAIN IT, YOU CAN ONLY EXPLAIN

IF THIS LIGATION EVENT COME FROM

THE TWO HOMOLOGUES BEING PRAIRD.

SO FOR EXAMPLE IF YOU GO BACK TO

THE DATA PUBLISHED IN HUMAN

CELLS YOU DO NOT SEE THAT.

SO IT COULD BE WE DID SOMETHING

HERE WHEN WE CONSTRUCTED THE

LIKLIBRARY OR IT COULD BE
TELLING

US THOSE ARE REGIONS OF THE

HOMOLOGOUS CHROMOSOME WHERE THE

PAIRING IS TAKING PLACE.

SO WE ANALYZE THE PAIRING SITES.

THEY ARE SHORT IN LENGTH.

THEY HAVE ALL KINDS OF CHROMATIN

AND ALL KINDS OF GENE ACTIVITY

AND THEY ARE ENRICHED, THEY ARE

DIVIDE OF LAMIN AND POLYCON AND

THEY ARE ENRICHED FOR EVERY

OTHER PROTEIN.

SO WE DON'T SPECIFICALLY SEE

INSULATOR PROTEINS IN THEM.

THEY ARE LOCATED AGAIN AT THE

BORDERS BETWEEN MODULES AND

INTERMODULES.

NOT SPECIFICALLY AT INTERMODULES

OR MODULES BUT AS A BOUNDARY.

AND I TOLD YOU THAT THOSE WERE

ALSO THE BOUNDARIES THAT AND

THERE ARE A FEW ONLY PER

CHROMOSOME.

IT'S A VERY SPARSE NUMBER OF

SITES THAT ARE KEEPING THE TWO

HOMOLOGUES TOGETHER.

AND THEY MUST BE THE SAME TYPE

OF OR A SUBSET OF INTERACTIONS

THAT ARE CAUSING THE

INTERACTIONS WITHIN THE

CHROMOSOME.

THEY ARE ALSO RESPONSIBLE FOR

KEEPING THE TWO HOMOLOGUES

TOGETHER.

SO IN THE LAST FEW MINUTES, I

WANT TO GO INTO THE IDEA THAT

THIS STRUCTURE THAT I JUST

DESCRIBED CAN BE ALTERED IN A

WAY THAT CAN BE REGULATED.

SO WHAT I TOLD YOU IS THAT THE

CHROMOSOME IS ORGANIZED INTO

MODULES AND INTERMODULES IN A

HIERARCHICAL FASHION.

AND THAT THE GENES THAT ARE IN

INTERMODULES ARE TRANSCRIBED AT

HIGH LEVELS.

SO THE OBVIOUS QUESTION IS TO

ASK HOW DOES THAT CHANGE FROM

CELL TYPE TO CELL TYPE.

HOW DOES IT CHANGE DURING

DIFFERENTIATION.

WHAT WOULD IT LOOK LIKE.

WHAT DO THE CHROMOSOMES LOOK

LIKE IN A STEM CELL AND THEN IF

YOU INDUCE THE STEM CELL TO

DIFFERENTIATE HOW DOES THAT

CHANGE.

IS IT DIFFERENT IN ONE CELL TYPE

VERSUS THE OTHER OR IT'S JUST A

STRUCTURE THAT IS COMMON TO ALL

THE CELLS AND REALLY WHAT IS

CONTROLLING GENE EXPRESSION IS

NOT THE STRUCTURE BUT THE

GENETIC MODIFICATIONS.

BUT I HOPE THAT THE ARGUMENTS

THAT I MADE CONVINCE YOU THAT

THAT'S NOT THE CASE AND THAT

THIS THREE DIMENSIONAL

ORGANIZATION IS SUPERIMPOSING

ANOTHER LEVEL OF REGULATION ON

THE GENETIC MODIFICATIONS OF THE

10 MAN OW NANOMETER FIBER.

SO WHAT WE ARE TRYING TO DO IS

TRY TO SEE IF THIS ORGANIZATION

IS DIFFERENT BETWEEN DIFFERENT

CELL TYPES.

I ALSO TOLD YOU THAT THE

INTERACTIONS RESPONSIBLE FOR

CREATING DISORGANIZATION

APPEARED TO BE MEDIATED IN LARGE

PART BY INSULATOR PROTEINS.

SO IF WE LOOK AT THE

DISTRIBUTION OF INSULATOR

PROTEINS IN DIFFERENT CELL

TYPES, WE SEE THAT AT LEAST IN

DROSPHILA THERE IS SOME

DIFFERENCES.

AND THERE'S ALSO DIFFERENCES IN

VERTEBRATES AND IT'S AN ISSUE OF

WHETHER YOU BELIEVE THAT THE

DIFFERENCES ARE REAL OR THEY ARE

A CONSEQUENCE OF JUST NOISE IN

THE CHIP C EXPERIENCE.

BUT IN DROATIO DROSPHILA WE KNOW
WE CAN

REGULATE INSULATOR ACTIVITY

EITHER BY CHANGING THE

RECRUITMENT OF THE DNA BINDING

PROTEIN OR CHANGING THE

RECRUITMENT OF THIS PROTEIN

CALLED PC190 THAT BINDS TO THE

DNA BINDING PROTEIN.

SO WE KNOW WE CAN FIND

DIFFERENCES BETWEEN DIFFERENT

CELL TYPES IN THAT TYPE OF

ARRANGEMENT.

SO HOW COME WE REGULATE THIS

INTERACTIONS.

SO WE'VE BEEN LOOKING AT ONE

SPECIFIC MODIFICATION AND THAT

IS PI PYLORATION.

IN BOTH IN VIVO, IT CAN BE PIE

LOWERRATION, IT AFFECTS THE

ABILITY OF THIS PROTEIN TO

INTERACT.

SO I THINK I HAVE THAT IN A

SLIDE PARROT ON.

SO THE ABILITY OF CP190 AND CPCF

TO INTERACT IS REGULATED BY

PYLORATION.

AND THIS EFFECT INSULATOR

ACTIVITY.

AND WE MEASURE INSULATOR

ACTIVITY BY AN ARCANE METHOD

THAT INVOLVES LOOKING AT

PHENOTYPES OF FLIES.

SO THIS IS A GENE WITH A

PROMOTER AND ENHANCER.

NORMALLY THE GENE IS EXPRESSED

AND MAKE THE BLACK HOLE OF THE

ABDOMEN.

IF WE HAVE INSULATOR BETWEEN THE

ENHANCER AND PROMOTER THEN THE

GAIN IS MUTANT.

SO IF WE MUTATE AND THIS IS

SOMETHING, IF WE MUTATE PARP

WHICH IS THE ENZYME, THEN THE

FLIES DIE.

SO WE CAN'T LOOK AT ANYTHING.

SO WHAT WE DO IS HAVE, LOOK AT

THE EFFECT OF JUST HETEROZYGOUS

MUTATION.

I DON'T KNOW IF YOU CAN SEE IT,

BUT BY MUTATING PARP, THE

INSULATOR CANNOT WORK AND THEN

THE BODY COLOR OF THE FLY IS

BLACK.

SO IN VIVO LACK OF PYLORATION

AFFECTS INSULATOR ACTIVITY.

THIS IS ANOTHER INSULATOR THAT

IS CONTROLLED BY CTCF IF WE

MUTATE PARP THEN THE EYES TURN

RED THAT MEANS THE INSULATOR IS

NOT WORKING SO WELL.

SO THIS IS THE SLIDE THAT SHOWS

YOU THAT IF WE INHIBIT

PYLORATION THEN CP190, CTCF

CANNOT INTERACT WITH CP190.

SO ALL THIS SORT OF SUGGESTS

THAT PIE HERRATION IS REGULATING

ACTIVITY BY REGULATING THE

INTERACTION BETWEEN THESE TWO

PROTEINS AND WE KNOW THE

INTERACTIONS BETWEEN THE TWO

PROTEINS ARE IMPORTANT FOR

INSULATOR FUNCTION.

SO IF HE F WE LOOK AT THE

GENOMICS AT THIS PROTEIN YOU

WOULD ASSUME IF WE INHIBIT

PYLORATION PI BY TREATING WITH A

DRUG CALLED 3AB THEN THE

LOCALLATION THE GENOME-WIDE

DISTRIBUTION OF THESE PROTEINS

WILL CHANGE.

AND THAT IS TRUE MANY SITES

ACTUALLY MOST OF THE SITES ARE

NOT AFFECTED.

BUT THERE ARE A SUBSET OF SITES

THAT ARE DRAMATICALLY REVIEWED

OR COMPLETELY GONE WHEN WE

INHIBIT PYLORATION.

THIS IS THE NUMBER OF SITES THAT

CHANGE OR ALL OF THE CHANGES.

THERE ARE TESTS WE CAN DO

WHETHER THIS IS AFFECTING OR

NOT.

THE OTHER TEST OF INSULATOR

ACTIVITY IS THE ABILITY OF THE

PROTEINS TO MAKE A LOOP.

ONE OF WAYS WE LOOK AT THAT IS

BY LOOKING AT CELLS.

SO IN CELL IN THE NORMAL CELLS

IF WE LOOK AT THE DISTRIBUTION

OF INSULATOR PROTEINS THEY TEND

TO BE IN THE PERIPHERY OF THE

NUCLEUS NEXT TO LAMIN AND THEY

FORM THIS STUFF THAT WE CALL

INSULATOR BODIES.

AND ALTHOUGH WE'VE NEVER

DEMONSTRATED SPECIFICALLY THIS

WE THINK THIS INSULATOR BODIES

ARE PLACES WHERE THE DIFFERENT

INSULATOR ARE COMING TOGETHER

AND INTERACTING AS SPECIFIC

REGION OF THE NUCLEUS.

IF WE HAVE THE CELLS WITH THIS

INHIBITOR OF PIRATION, YOU CAN

SEE THE INSLAIRLT PROTEINS ARE

STILL THERE BUT NOW DIFFUSED

THROUGH THE NUCLEUS.

ON THE BASIS OF THIS CRITERIA IT

LOOKS LIKE INAWE BUSINESS OF

PYLORATION AFFECTS THE INSULATOR

PROTEINS WHICH ARE BOUND TO

CERTAIN REYNOLDS OF THE NUCLEUS

TNUCLEUS -- SERD REGIONS OF THE

NUCLEUS.

WHAT WE'VE DONE IS 4C.

WE'VE USED ONE OF THESE SITES

THAT IN WHICH INHUH LITTLE OF

PIRATION AND WE STUDY

INTERACTIONS BY 4C WITH OTHER

REGIONS OF THE GENOME AND THEN

WE'VE GONE SPECIFICALLY TO THOSE

REGIONS AND QUANTITATIVE

INTERACTION.

YOU CAN SEE THOSE REGIONS THAT

INTERACTION WITH THESE BASS

WHICH C 190, IN THOSE REGIONS

THE INTERACTION BETWEEN THE BASE

AND OTHER REGIONS THROUGH THE

KNEE JUST A MOMENT GOES DOWN.

SO PARYLATION AFFECTS WHAT IS

NECESSARY TO FORM A LOOP.

THIS WOULD BE AN ACTIVE

INSULATOR THAT IS FORMING A

LOOP.

THIS IS AN INACTIVE INSULATOR

THAT'S UNABLE TO FORM A LOOP.

PARYLATION IS REQUIRED FOR THIS

INTERACTION.

AND IF YOU INHIBIT PARYLATION AT

A SUBSET OF SITES IN THE GENOME,

THERE IS A DECREASE OF

INTERACTION AND ALSO IMPAIRMENT

OF INSULATOR FUNCTION IN VIVO AS

CHARGED BY THE PHENOTYPE OF THE

FLIES THAT I TOLD YOU.

SO I'M GOING TO ADDRESS ONE MORE

QUESTION.

AND THAT IS WHAT HAPPENS TO THE

THREE DYE MENTIONAL ARCHITECTURE

OF THE NUCLEUS DURING THE CELL

CYCLE.

AND THIS IS A QUESTION FOR WHICH

I DON'T HAVE AN ANSWER.

I'M JUST GOING TO GIVE YOU SOME

VERY PRELIMINARY RESULTS OF THE

THINGS THAT WE ARE DOING THE

LAST THREE OUR FOUR MINUTES.

SO WHEN WE LOOK AT THE DIFFERENT

INSULATOR PROTEINS TO SEE WHAT

HAPPENS DURING THE CELL CYCLE,

DURING MITOSIS, THE ARCHITECTURE

OF THE CHROMOSOME CHANGES

DRAMATICALLY.

AND SO IF INSULATOR PROTEINS ARE

REGULATING THIS ARCHITECTURE,

THEN SOMETHING, THEY MUST BE

CHANGING AND THEY MUST BE

REARRANGING AND THEY MUST BE

DOING THINGS TO ALLOW THE

TRANSITION FROM INTERFACE

CHROMATIN STRUCTURE TO MITOTIC

CHROMATIN STRUCTURE.

SO IF WE LOOK AT THE

DISTRIBUTION OF THESE PROTEINS,

WE SEE THAT IN MITOSIS, CP190

TENDS TO DISAPPEAR FROM THE

CHROMOSOMES AND IT'S LOCATED IN

THE PERIPHERY OF THE M MITOTIC

PLATE.

I DON'T KNOW IF YOU CAN PRESSURE

IT HERE BUT CTCF DOES THE SAME

THING WHERE A MODIFIER JUST GETS

DIFFUSED THROUGH THE NUCLEUS,

THEY DON'T FOLLOW THE SAME

PATTERN.

SO WE LOOK AT THE DISTRIBUTION

OF THESE PROTEINS BY CHIP C

COMPARING INTERFACE LOCALIZATION

WITH LOCALIZATION IN METAPHASE

CHROMOSOME.

IT'S ALMOST IMPOSSIBLE TO

SYNCHRONIZE CELLS SO WHAT WE

HAVE TO DO WAS JUST A POPULATION

OF GROWING CELLS AND SORT THEM

TO SPECIFICALLY ISOLATE THE

POPULATION OF M MITOTIC CELLS.

WHAT YOU CAN SEE HERE WHICH YOU

DON'T HAVE TO EXAMINE IN DETAIL

AND I'LL JUST TELL YOU IS THAT

SOME OF THE PROTEINS CHANGED

DRAMATICALLY DURING MITOSIS.

SO THERE'S ONLY, THERE ARE A

THOUSAND SPEAKS BU PEAKS BUT
THOSE PEAKS

ARE ALMOST UNDETECTABLE.

AND I HAD A SLIDE TO SHOW YOU

THAT.

SO WHAT HAPPENED IS THAT IT

ESSENTIALLY LEAVES THE

CHROMOSOME AND CTCF AND CP   190

VERY A LARGE NUMBER OF NEW SITES

IN MITOSIS.

THEY ARE SPECIFIC FOR MITOSIS.

THEY WERE NOT THERE IN

INTERFACE.

IF THEY ARE MEDIATING

INTERACTION.

THIS TELLS US IN THE

REORGANIZATION OF THE

CHROMOSOMES BETWEEN INTERFACE

AND MITOSIS, THAT, MAYBE THAT

REORGANIZATION OF THE CHROMOSOME

IS MEDIATED BY INSULATOR

PROTEINS.

IF WE LOOK TO SEE WHERE THOSE

NEW SITES ARE WHEN WE COMPARE

INTERFACE WITH MITOSIS, WHAT WE

SEE IS THAT THE NEW SITES ARE

PRESENT IN HIGHLY TRANSCRIBED

GENES.

OKAY.

SO THERE'S A SCRIPT FROM -- LAST

RECENTLY THAT SHOWED VERY NICELY

THAT COMPONENTS, THE MSL-3

PROTEIN WHICH IS A PROTEIN THAT

IS A COMPONENT OF THE DOSAGE

COMPENSATION PATHWAY FOR

DROSPHILA.

NOW THAT PROTEIN STAYS

ASSOCIATED WITH CHROMOSOMES IN

MITOSIS AND THAT IS PRESENT ON

THE SURFACE OF THE METAPHASE

CHROMOSOME.

AND THE IDEA IS THAT THAT

PROTEIN IS THERE TO KEEP THOSE

GENES THAT NEED TO BE HIGHLY

EXPRESSED OR EXPRESSED

IMMEDIATELY AFTER G1 TRANSITION

TO KEEP THOSE GENES IN A SPECIAL

ARRANGEMENT ON THE MELT AWE --

METAPHASE CHROMOSOME.

WE COMPARED THE DISTRIBUTION OF

THE PROTEINS WITH CP190 OR WITH

CPCF AND WHAT WE SEE IS DURING

MITOSIS, THESE TWO PROTEINS

ACTUALLY LOCALIZE WITH MSL2, FOR

EXAMPLE.

SO ALTHOUGH THESE RESULTS ARE

PRELIMINARY, IT SUGGESTS TO US

THAT INSULATOR PROTEINS MAYBE

ARE INVOLVED IN CREATING CERTAIN

ARRANGEMENT OF THE MY TAUGH
MITOTIC

CHROMOSOMES TO MAKE SURE THAT

EITHER THOSE GENES ARE ACTIVE,

SO THAT GENES STAY ON THE

SURFACE OF THE CHROMOSOME AND

THOSE GENES ARE ACTIVATED

QUICKLY SO THEY ARE BOOK MARKED

FOR RAPID ACTIVATION OR ANOTHER

FORM OF REGULATION JUST BECAUSE

THE INSULATOR PROTEINS AT THE

NEW SITES SEEM TO BE ASSOCIATED

WITH HIGHLY TRANSCRIBED GENES.

SO THAT INSULATOR PROTEINS WOULD

REGULATE A TRANSITION THAT GOES

FROM INTERFACE FORMING

STRUCTURES LIKE THIS TO MITOSIS

IN WHICH THIS IS THE MITOTIC

CHROMOSOME.

THEY ARE KEPT ON THE SURFACE TO

MAINTAIN CERTAIN GENES IN MORE

OPEN CONFIRMATION.

SO I'M GOING TO FINISH HERE, I

JUST WANTED TO LEAVE YOU WITH A

FEW TAKE HOME MESSAGES ONE IS

THAT WE BELIEVE AND WE ARE

GETTING MORE EVIDENCE EVERY DAY

THAT THE GENOME IS ORGANIZED IN

THIS COMPLEX HIERARCHICAL THREE

DIMENSIONAL STRUCTURE THAT IS

MADE OUT OF THIS MODULES AND

INTERMODULES.

AND I THINK THIS IS GOING TO BE,

SO I NEVER MADE THE CONNECTION

TO THE POLYTIN CHROMOSOME BUT WE

ARE TRYING VERY HARD TO SEE IF

THE MODULES IN INTERFACE

CHROMOSOME ARE THE BONDS THAT

DARK REGIONS IN MALL CONTINUE

CHROMOSOMES AND -- POLYTIN

CHROMOSOMES.

AND THIS STRUCTURE CARRIES THE

GENETIC INFORMATION.

WE DON'T HAVE EVIDENCE FOR THAT

BUT MAYBE CELL TYPE SPECIFIC BUT

WE DO THINK BASED ON THE RESULTS

WE HAVE UNTIL NOW THAT THAT, THE

MODULE VERSUS INTRAMODULE

STRUCTURE CARRIES ANOTHER LEVEL

OF REGULATION THAT IS

SUPERIMPOSED ON THE PRIMARY

GENETIC MODIFICATIONS ON THE

NANOMETER FIBER.

SOME OF THESE INTERACTIONS MAY

BE CAUSAL AND SOME OF THEM MAY

JUST BE AN EFFECT.

SO I THINK THERE'S A VERY DITCH

VIEW OF THE STRUCTOR -- VERY

DIFFERENT VIEW OF THE STRUCTURE

OF THE CHROMOSOME IF YOU THINK

THERE ARE SOME PROTEINS THAT ARE

THERE TO CREATE A STRUCTURE BUT

THEN ALLOWS A CERTAIN FUNCTIONAL

OUTPUT VERSUS FUNCTION IN TERMS

OF TRANSCRIPTION DRIVING THE

STRUCTURE.

SO THOSE ARE TWO VERY OPPOSITE

VIEWS OF WHAT IS HAPPENING TO

THE CHROMOSOME, AND I THINK

PROBABLY THE TRUTH IS SOMEWHERE

IN BETWEEN.

WE WOULD LIKE TO EVENTUALLY SHOW

THAT THIS THREE DIMENSIONAL

STRUCTURE, AND WE WILL KNOW WHEN

WE LOOK AT MORE CELL TYPES IS

SORT OF A FINGERPRINT OF

IDENTITY.

SO THE DIFFERENT CELLS WOULD

HAVE A DIFFERENT STRUCTURE BUT

IT'S ALSO A BLUE PRINT OF WHAT

THE FUNCTIONAL OUTPUT ON THE

GENOME IS.

IN OTHER WORDS HOW THE GENOME

CAN BE INTERPRETED AND

PRESCRIBED.

SO LET ME JUST FINISH BY

ACKNOWLEDGING THE PEOPLE THAT

DID THE WORK, ALL THE HIGH C

EXPERIMENTS WERE DONE BY -- WHO

IS A VERY TALENTED POST DOC IN

MY LAP.

THE PARYLATION STORY I TOLD YOU

IS THE WORK OF ANOTHER POST DOC.

AND THE MITOSIS STUFF IS

[INDISCERNIBLE] SO THANK YOU

VERY MUCH.

[APPLAUSE]

>> WE CAN TAKE A FEW QUESTIONS

IF YOU WOULD LIKE.

PLEASE ADDRESS YOUR QUESTION AT

THE MICROPHONES THOSE OF YOU WHO

MAY.

YES, SIR.

>> SO VERY NICE.

THIS EVIDENCE OF REGULATION,

THERE'S A FAIRLY VAST LITERATURE

ABOUT REGULATIONS ITSELF.

HOW DO YOU ENVISION THAT KIND OF

REGULATION WORKING

PHYSIOLOGICALLY?

>> I HAVEN'T THOUGHT ABOUT THAT.

YOU MEAN WHAT IS REGULATING

PARP, HOW IS PARP TARGETED TO

SPECIFIC SITES.

>> UNDER WHAT PHYSIOLOGIC

CONDITIONS WOULD SUCH STRUCTURES

BE PHYSIOLOGICALLY RELEVANT.

>> I DON'T HAVE AN ANSWER TO

THAT REALLY.

IT'S PROBABLY A VERY COMPLICATED

ANSWER IN VIEW OF WHAT WE KNOW

ABOUT PARP IN VERTEBRATES AND

HOW IT AFFECTS ALL KINDS OF

THINGS.

SO I DON'T KNOW YET.

UNTIL NOW, THE ONE THING THAT

HAS BEEN BOTHERING ME MOST IS

HOW DOES PARP RECOGNIZE SOME

INSULATOR SITES BUT NOT OTHERS.

HOW IS IT DECIDING BETWEEN THAT.

AND WE DON'T HAVE AN ANSWER.

>> SO I HAVE A QUESTION

REGARDING THE RELATIONSHIP, THE

PHYSICAL RELATIONSHIP BETWEEN

THE DOMAINS AND INTERDOMAINS AND

THE TRANSCRIPTIONAL STATUS OF

THE BLOCK.

IF YOU DISRUPT HISTO

MODIFICATIONS OR DEPLETING HISTO

MET OWE TRANSFER ACES, DOES THAT

AFFECT THE DOMAIN STRUCTURES

NOW.

>>> I WOULD SAY NO.

DO YOU MEAN GLOBALLY IN THE

WHOLE GENOME?

>> RIGHT.

SOME OF THE DOMAINS WERE

SPECIFICALLY THE INTERDOMAINS, A

LOT OF INTERACTIONS ARE RELATED

TO TRANSCRIPTION AND RNA

POLYMERASE.

WE DON'T KNOW IF RNA PLAIN RACE

IRACE -- PRELIMINARY RACE IS --
POLYMERASE
.

I WOULD SAY IF YOU INHIBIT TRY

METHYL, I THINK YOU WOULD AFFECT

THE DOMAIN JUST BECAUSE YOU

COULD AFFECT ALL KINDS OF

INTERACTIONS AND YOU WOULD

AFFECT THE FOLDING.

>> THE GREEN -- THAT YOU SHOW IS

THAT [INDISCERNIBLE]

>> I DON'T KNOW.

THAT'S A GOOD QUESTION.

I DON'T THINK THAT HAS BEEN

DONE, HAS BEEN ADDRESSED, YES.

BUT I THINK THAT WOULD BE AN

IMPORTANT THING TO DO, YES.

>> MANY OF THE DROSPHILA LINES

HAVE AN IO ENDOGENOUS

[INDISCERNIBLE] AND FIVE, SIX

YEARS AGO THEY FOUND THE ENTIRE

GENOME IS EMBEDDED IN THE

DROSPHILA GENOME AS WELL.

SO MAPPING THIS SUBSET OF GENES

ON TO YOUR MODULAR, INTERMODULAR

MAP COULD BE VERY INTERESTING.

SINCE THEY CLEARLY WORK TOGETHER

TO MODIFY THEY HAD INTRACELLULAR

ORGANISM.

>> SO IF THE JEES GENOME OF THIS

ORGANISM INTEGRATED INTO THE --

GENOME.

>> YES, IT IS.

>> IN LOT OF DIFFERENT PLACES.

>> THAT'S WHAT I'M ASKING YOU.

[LAUGHTER]

YOU COULD BASICALLY GET A LINE

AND LOOK AT IT.

YOU CAN RIGHT NOW, THE GENES ARE

THERE AND YOU'VE GOT THEM AND

YOU COULD MAP THEM ON TO YOUR

MODULAR MAP AND JUST SEE WHERE

THEY END UP.

>> I MEAN THOSE ARE, THAT'S THE

INFORMATION THAT WE GET RID OF

FIRST.

>> RIGHT.

A LOT OF PEOPLE GET RID OF THE

INTERESTING STUFF.

[LAUGHTER]

>> SO THERE'S A LOT OF

INFORMATION THAT WE ELIMINATED.

FOR EXAMPLE THE NUCLEUS, WE GET

RID OF IT.

>> IT BASICALLY SHEDS ITS GENOME

BECAUSE IT DOESN'T NEED TO HAVE

IT LIKE A CAR ON SMOOTH ROADS

WILL GET RID OF ITS SPARE TIRE.

BUT YOU CAN CLEARLY, BECAUSE

THERE ARE FREE LIVING

WELLBACHIA, YOU KNOW EXACTLY

WHAT THE GENES ARE, YOU COULD GO

BACK TO WHAT YOU THREW AWAY AND

GO THROUGH THE WELLBACHIA AND

MAP THEM ON.

THAT WOULD BE FASCINATING.

>> THANK YOU.

I THINK THAT'S A GOOD IDEA.

>> OKAY.

VICTOR HAS AN AIRPLANE TO CATCH

SO I THINK WE SHOULD CLOSE.

THANKS AGAIN FOR A VERY

INTERESTING PRESENTATION.

[APPLAUSE]

AND REMEMBER THE RECEPTION

IMMEDIATELY ADJACENT HERE IN THE